WO2011112245A1 - Analyse par liaison à la luciférase de l'adn-méthyltransférase, de la protéine méthyltransférase et de la s-adénosylhomocystéine et leurs applications - Google Patents

Analyse par liaison à la luciférase de l'adn-méthyltransférase, de la protéine méthyltransférase et de la s-adénosylhomocystéine et leurs applications Download PDF

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WO2011112245A1
WO2011112245A1 PCT/US2011/000430 US2011000430W WO2011112245A1 WO 2011112245 A1 WO2011112245 A1 WO 2011112245A1 US 2011000430 W US2011000430 W US 2011000430W WO 2011112245 A1 WO2011112245 A1 WO 2011112245A1
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methyltransferase
adohcy
homocysteine
protein
enzyme
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Vern L. Schramm
Ivan Hemeon
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Albert Einstein College Of Medicine Of Yeshiva University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to methods for detecting DNA (cytosine 5)- methyltransferase activity and protein methyltransferase activity, and for screening for inhibitors of DNA (cytosine 5)-methyltransferase and protein methyltransferase.
  • the invention also relates to detecting S-adenosylhomocysteine and screening inhibitors for enzymes that form S-adenosylhomocysteine as a product of the reaction.
  • AdoMet methyltransferases catalyze the transfer of a methyl group from 5- adenosyl-l-methionine (AdoMet) to a methyl acceptor.
  • Methyl acceptors include proteins, lipids, carbohydrates, oligonucleotides, and various small molecule metabolites.
  • cytosine 5)-methyltransferases (cytosine 5)-methyltransferases (DNMTs, EC 2.1.1.37) catalyze the transfer of a methyl group from AdoMet to a DNA substrate. This methyl group is transferred to the 5-position of a cytosine base when the cytosine precedes a guanosine residue at so-called "CpG islands" and effectively inhibits transcription of the associated gene (1 ).
  • DNMTs are an important part of the mechanisms by which gene transcription is controlled in development, differentiation and cancer.
  • a scintillation proximity assay avoids separation of AdoMet and DNA since DNA binds to yttrium silicate scintillant beads, but this approach also suffers from nonspecific AdoMet binding (1 1 ).
  • Fluorescence-based assays use an anti-AdoHcy antibody in conjunction with AdoHcy bearing a fluorescent tag (12), or use 5-adenosyl-l- homocysteine hydrolase (SAHH) to generate homocysteine that reacts with thiol-sensitive fluorophores (13, 14). These assays do not have a large range and require the use of thiol-free assay mixtures or the use of anaerobic conditions (13).
  • Spectrophotometric assays have been developed for other AdoMet-dependent methyltransferases, one using SAHH and Ellman's reagent under discontinuous anaerobic conditions (15), and another using 5-adenosyl-l- homocysteine nucleosidase to generate adenine from AdoHcy hydrolysis which is then converted to hypoxanthine using adenine deaminase (16). These cannot accommodate low AdoMet or DNA concentrations due to low sensitivity and also encounter interfering absorption from DNA substrates.
  • the present invention addresses the need for an improved DNMT assay, which can be used to screen for inhibitors of DNMT.
  • the assay converts S- adenosylhomocysteine to a luciferase-based light signal and is suitable for application to any enzymatic reaction that forms S-adenosylhomocysteine as a product of an enzymatic reaction.
  • the present invention provides methods of detecting the presence of DNA (cytosine 5)-methyltransferase (DNMT) or a protein methyltransferase (PMT) comprising: (a) enzyme-catalyzed transfer by DNMT or the PMT of a methyl group from 5-adenosyl-l- methionine (AdoMet) to a DNA substrate or to a protein to produce S-adenosyl-l- homocysteine (AdoHcy); (b) hydrolyzing AdoHcy to adenine by 5'-methylthioadenosine nucleosidase (MTAN); (c) converting adenine to adenosine 5 '-monophosphate (AMP) by adenine phosphoribosyl transferase (APRTase) in the presence of 5-phospho-a-drribosyl-l - pyrophosphate (PRPP); (d)
  • the invention further provides methods of determining whether or not a compound is an inhibitor of DNA (cytosine 5)-methyltransferase (DNMT) or an inhibitor of a protein methyltransferase (PMT) comprising carrying out any of the methods disclosed herein for detecting the presence of DNMT or PMT in the presence of the compound and in the absence of the compound, wherein a decrease in the luminescent signal in the presence of the compound is indicative that the compound is an inhibitor of DNMT or PMT, and wherein a lack of decrease in the luminescent signal in the presence of the compound is indicative that the compound is not an inhibitor of DNMT or PMT.
  • DNA cytosine 5)-methyltransferase
  • PMT protein methyltransferase
  • the assay of the present invention converts S-adenosylhomocysteine to a luciferase-based light signal and is suitable for application to any enzymatic reaction that forms S-adenosylhomocysteine as a product of an enzymatic reaction.
  • FIG. 1 Schematic overview of the AdoHcy detection assay.
  • AdoHcy is formed from AdoMet-dependent DNA methylation via DNMT and is converted to adenine using MTAN.
  • the adenine is converted to AMP in an APRTase-catalyzed reaction with PRPP, then to ATP using PPD with phosphoenolpyruvate.
  • the ATP then reacts with luciferin and 0 2 in a firefly luciferase-catalyzed process to generate a stable light signal for quantification as the resulting AMP is continuously cycled to ATP.
  • FIG. 2A-2B AdoHcy standards: (A) 36 pmol AdoHcy in 25 ⁇ , DNMT assay buffer and 25 ⁇ , continuous assay buffer measured in the kinetic acquisition mode of the luminometer at room temperature. Signal reached maximum value within 90 s. (B) AdoHcy standard curve obtained by mixing 25 ⁇ , AdoHcy solutions in DNMT assay buffer (0.24-800 pmol) with 25 ⁇ , continuous assay buffer, incubating at room temperature for several minutes, and then measuring luminescence in the discontinuous format.
  • Figure 3A-3B Continuous assay measurements on 4 U M.SssI CpG methyltransferase (45 nM) and 1 ⁇ g poly(dldC) at room temperature: (A) Reaction time course with increasing AdoMet concentrations. (B) Initial rates fit to Michaelis-Menten equation.
  • FIG. 4A-4E Continuous assay measurements on human DNMTs at room temperature. Initial rates fit to Morrison equation for tight binding substrates: (A) 1 ⁇ g poly(dldC), 492 nM DNMT3L-CD-DNMT3b, varied AdoMet concentration. (B) 10 ⁇ AdoMet, 492 nM DNMT3L-CD-DNMT3b, varied poly(dldC) concentration. (C) 10 ⁇ AdoMet, 490 nM DNMT3 L-CD-DNMT3 a, varied poly(dldC) concentration. (D) 1 ⁇ g poly(dldC), 516 nM DNMT1 , varied AdoMet concentration. (E) 35 ⁇ AdoMet, 516 nM DNMT1 , varied poly(dldC) concentration.
  • Figure 5A-5B Initial rates for 258 nM DNMT1, 6 ⁇ g poly(dldC), 300 ⁇ , total volume at room temperature and at 37 °C measured by discontinuous assay: (A) 682 nM AdoMet, luciferase-linked AdoHcy assay. (B) 672 nM [methyl- 3 U] -AdoMet, radiometric filter paper assay.
  • the invention provides a method of detecting the presence of DNA (cytosine 5)-methyltransferase (DNMT) or a protein methyltransferase (PMT) or any enzyme that forms S-adenosyl-l-homocysteine (AdoHcy) as a product comprising: (a) enzyme-catalyzed transfer by DNMT or PMT of a methyl group from S-adenosyl- 1-methionine (AdoMet) to a DNA substrate or to a protein to produce S-adenosyl-1- homocysteine (AdoHcy), or enzyme-catalyzed transfer by any enzyme that forms iS-adenosyl- 1-homocysteine (AdoHcy) as a product;
  • APRTase phosphoribosyl transferase
  • PRPP 5-phospho-a-d-ribosyl-l- pyrophosphate
  • the presence of the luminescent signal indicates the presence of DNMT or the
  • step (e) The AMP that is produced in step (e) can be used as a source of AMP in step (d).
  • MTAN is present in a concentration between 5 ⁇ and 125 nM.
  • Luciferase is a common bioluminescent enzyme. Organisms from which luciferase can be obtained include, but are not limited to fireflies, bacteria (e.g. Vibrio fischeri), jellyfish (e.g. Aequorea victoria), beetles, and squid. In the preferred embodiment of the present invention, the luciferase is firefly luciferase.
  • the luminescent signal that is produced has an intensity that is proportional to the amount of DNMT or PMT or the enzyme that forms S-adenosyl-1- homocysteine (AdoHcy) as a product over a range of 0.1-1000 pmol AdoHcy.
  • AdoHcy S-adenosyl-1- homocysteine
  • maximum luminescence is reached within 2 minutes.
  • DNMT or PMT or the enzyme that forms S-adenosyl-l-homocysteine
  • (AdoHcy) as a product can be detected in a continuous assay or in a discontinuous assay.
  • Examples of PMT that can be detected include, but are not limited to, protein arginine N-methyltransferase, protein-glutamate O-methyltransferase, protein-histidine N- methyltransferase, protein-S-isoprenylcysteine O-methyltransferase, myelin basic protein- arginine N-methyltransferase, protein L-isoaspartyl methyltransferase, protein-S- isoprenylcysteine O-methyltransferase, and an enzyme that forms S-adenosyl-l-homocysteine (AdoHcy) as a product.
  • protein arginine N-methyltransferase protein-glutamate O-methyltransferase
  • protein-histidine N- methyltransferase protein-S-isoprenylcysteine O-methyltransferase
  • Methyl acceptors include proteins, lipids, carbohydrates, oligonucleotides, and various small molecule metabolites.
  • Examples of enzymes that form S-adenosyl-L-homocysteine (AdoHcy) as a product include, but are not limited to, histamine N-methyltransferase, phenylethanolamine N-methyltransferase, tryptamine N-methyltransferase, phosphatidylethanolamine N- methyltransferase, O-5-hydroxyindole-O-methyltransferase, acetylserotonin O- methyltransferase, catechol-O-methyl transferase, homocysteine betaine-homocysteine methyltransferase, homocysteine methyltransferase, phosphatidyl ethanolamine
  • the invention also provides a method of determining whether or not a compound is an inhibitor of DNA (cytosine 5)-methyltransferase (DNMT) or an inhibitor of a protein methyltransferase (PMT) or an inhibitor of an enzyme that forms 5-adenosyl-l- homocysteine (AdoHcy) as a product, comprising carrying out any of the methods disclosed herein for detecting the presence of DNMT or PMT or an enzyme that forms 5-adenosyl-l- homocysteine (AdoHcy) as a product, in the presence of the compound and in the absence of the compound, wherein a decrease in the luminescent signal in the presence of the compound is indicative that the compound is an inhibitor of DNMT or PMT or the enzyme that forms S- adenosyl-l-homocysteine (AdoHcy) as a product, and wherein a lack of decrease in the luminescent signal in the presence of the compound is
  • a sensitive luciferase-linked continuous assay was developed that converts the S-adenosyl-L-homocysteine (AdoHcy) product of DNA methylation to a quantifiable luminescent signal.
  • the assay can be used under continuous or discontinuous conditions in high-throughput plate formats.
  • MTAN 5'-Methylthioadenosine nucleosidase hydrolyzes AdoHcy to adenine which is converted to adenosine 5 '-monophosphate (AMP) by adenine phosphoribosyl transferase (APRTase, EC 2.4.2.7) using 5-phospho-a-d-ribosyl-l- pyrophosphate (PRPP), then to adenosine 5 '-triphosphate (ATP) by pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1) using phosphoenolpyruvate (PEP) and inorganic pyrophosphate.
  • AMP adenosine 5 '-monophosphate
  • APRTase adenine phosphoribosyl transferase
  • PRPP 5-phospho-a-d-ribosyl-l- pyrophosphate
  • ATP pyruv
  • AdoHcy is an inhibitor of AdoMet-dependent methyltransferases (18), its removal by MTAN also prevents product inhibition. The continuous nature of this assay eliminates product separation and sample preparation steps.
  • the kinetic properties are characterized for two highly active DNMTs, human DNMT1 and the bacterial M.SssI CpG methyltransferase, and two DNMTs of low activity, complexes of human DNMT3L with the catalytic domains of human DNMT3a and
  • DNMT3b The full-length human DNMT1 is thought to be responsible for maintaining the methylation pattern of the genome during cell division and genome replication (1). DNMT3a and DNMT3b are thought to play roles in de novo methylation of the genome during embryonic development and have also been implicated in cancers (1). Activation was characterized by human DNMT3L, a catalytically inactive accessory protein that interacts with DNMT3a and DNMT3b to enhance their activities.
  • M.SssI CpG methyltransferase and AdoMet were purchased from New
  • AdoMet was further purified by reverse-phase HPLC using a Waters CI 8 column (4.6 x 250 mm), 20% acetonitrile at 0.5 mL/min.
  • Poly(2'- deoxyinosinic-2'-deoxycytidylic acid) was purchased from Sigma (Ashland, Massachusetts; Cat. No. P4929).
  • Firefly luciferase ATP assay kit (ATPlite) and Betaplate Scint scintillation fluid were purchased from Perkin-Elmer, as was S-adenosyl-l-[wet/2 , /- 3 H]methionine with a specific activity of 70.8 Ci/mmol (Waltham, Massachusetts).
  • EDTA-free Complete protease inhibitor cocktail tablets and PhosSTOP phosphatase inhibitor tablets were purchased from Roche Applied Science (Indianapolis, Indiana).
  • Recombinant S. cerevisiae APRTase bearing an N-terminal His tag and recombinant Clostridium symbiosum PPDK were prepared as previously described (17).
  • Synthetic DNA constructs coding for DNMTs and MTAN were purchased from DNA 2.0 (Menlo Park, California).
  • the Gateway LR Clonase II enzyme mix was purchased from Invitrogen (Carlsbad, California). Plasmid DNA was isolated and purified using the Qiagen HiSpeed plasmid midi kit (Valencia, California).
  • Ni-NTA agarose was purchased from Qiagen. Corning 96-well non-binding surface microplates were purchased through Fisher Scientific (Pittsburgh, Pennsylvania). Protein concentrations were measured by Bradford assay in comparison with bovine serum albumin (BSA) standards obtained from Bio-Rad (Hercules, California). All other reagents used were from Fisher Scientific or Sigma- Aldrich and were the highest quality available. Luminescence was measured using a Glomax 96-well luminometer from Promega (Madison, Wisconsin).
  • Human DNMT1 expression constructs containing an N-terminal His 6 tag and an rTEV protease cleavage site were subcloned from the pFastBac HTa plasmid (a generous gift of Prof. Keith D. Robertson, University of Florida).
  • Baculovirus Expression System manual (Invitrogen). The titer of recombinant baculovirus was measured by an endpoint dilution assay.
  • SF9 cells were grown in conical flasks at 120 rpm, 27 °C. SF9 cells at a density of 2 x 106 mL-1 were infected at a multiplicity of infection between 1 and 5.
  • the cells were harvested 96 hours post-infection, then lysed in RIPA buffer (PBS, 1 % NP-40, 0.5 % SDS, 0.5 % sodium deoxycholate, 10 % glycerol, Complete protease inhibitor cocktail tablet (Roche)) and were sonicated on ice 3 x 25 s using a Fisher Scientific model 500 sonicator at 20 % duty cycle. Cellular debris was removed by centrifugation at 16,000 rpm for 30 min and the cleared supernatant was loaded on a 1 mL HisTrap FF crude His-tag affinity column (GE Healthcare).
  • RIPA buffer PBS, 1 % NP-40, 0.5 % SDS, 0.5 % sodium deoxycholate, 10 % glycerol, Complete protease inhibitor cocktail tablet (Roche)
  • the column washed with buffer A (20 mM imidazole, 50 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) pH 7.4, 200 mM NaCl) and human DNMT1 was eluted with a gradient of 20- 500 mM imidazole in buffer A. Fractions were analyzed by SDS-PAGE and those containing human DNMT1 were combined and subjected to overnight dialysis in 50 mM HEPES pH 7.4, 200 mM NaCl and 2 mM dithiothreitol (DTT). The dialyzed human DNMT1 was concentrated to ⁇ 5 mg/mL using an Amicon ultracentrifugal filter device, 10,000 MW cut off (Millipore).
  • buffer A 20 mM imidazole, 50 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) pH 7.4, 200 mM
  • the plasmid was amplified in E. coli DH5a cells and purified by HiSpeed midiprep (Qiagen) to afford pDONR221 -CD-HDNMT3a.
  • the gene encoding CD-DNMT3a was cloned into the pDEST14 Gateway destination/expression vector using the Gateway LR Clonase II enzyme mix (Invitrogen) which was amplified in E. coli One Shot OmniMAX 2T1 phage-resistant cells (Invitrogen) and purified by HiSpeed midiprep to afford pDEST14-CD- HDNMT3a.
  • the expression vector was transformed into E.
  • the culture was cooled to 18 °C and expression was induced with 0.5 mM isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG). After incubating at 18 °C, 250 rpm for 15 h, the cells were harvested by centrifugation at 4,000 rpm for 20 min at 4 °C.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • the cell pellet was resuspended in 25 mL lysis buffer (100 mM HEPES pH 8.0, 300 mM NaCl, 10 mM imidazole, 5 % glycerol, 0.1 % DTT, EDTA-free Complete protease inhibitor cocktail tablet (Roche), 0.1 mg/mL DNasel, 0.1 mg/mL lysozyme) and passed through a French press (3 x 16,000 psi) followed by centrifugation at 16,000 g for 20 min at 4 °C.
  • lysis buffer 100 mM HEPES pH 8.0, 300 mM NaCl, 10 mM imidazole, 5 % glycerol, 0.1 % DTT, EDTA-free Complete protease inhibitor cocktail tablet (Roche), 0.1 mg/mL DNasel, 0.1 mg/mL lysozyme
  • the supernatant was loaded onto 2 mL Ni-NTA agarose resin (Qiagen) that had been equilibrated in running buffer (50 mM HEPES pH 8.0, 300 mM NaCl, 5 % glycerol).
  • running buffer 50 mM HEPES pH 8.0, 300 mM NaCl, 5 % glycerol.
  • the resin was washed with 30 mL running buffer supplemented with 100 mM imidazole and 0.1 % DTT, then CD-DNMT3a was eluted in running buffer supplemented with 500 mM imidazole and 0.1 % DTT.
  • the purity of the fractions was monitored by SDS-PAGE.
  • the first 3 mL 500 mM imidazole fraction was dialyzed against a storage buffer containing 20 mM HEPES pH 8.0, 250 mM NaCl, 5 % glycerol, 0.1 % DTT overnight at 4 °C and was then flash frozen in aliquots in dry ice/ethanol and stored at -80 °C. Protein concentration was determined by Bradford assay (Bio-Rad). Approximately 1.5 mg purified protein was obtained from 1 L of culture.
  • CD- DNMT3b Expression and Purification of Human DNMT3b Catalytic Domain
  • the catalytic domain for human DNMT3b was identified as described for CD- DNMT3a.
  • a construct coding for a 286 amino acid C-terminal domain of human DNMT3b (residues 568-853 of human DNMT3b, NCBI accession number Q9UBC3) containing an N- terminal His tag with a thrombin cleavage site was purchased from DNA 2.0 in a
  • pDONR221 Gateway donor vector (Invitrogen).
  • the gene was amplified (pDONR221 -CD- HDNMT3b), cloned into the pDEST14 Gateway expression vector, and amplified again (pDEST14-CD-HDNMT3b) as described for CD-DNMT3a.
  • the expression vector was transformed into E. coli BL21 (DE3) pLysS cells which were grown, induced, harvested, and lysed under the same conditions as described above.
  • CD-DNMT3b was purified and dialyzed under identical conditions as described above except that it was eluted with running buffer supplemented with 100 mM ethylenediaminetetraacetic acid (EDTA) and 0.1 % DTT and the second 3 mL fraction was dialyzed, then stored at -80°C. Approximately 2.2 mg purified protein could be obtained from 1 L of culture.
  • EDTA ethylenediaminetetraacetic acid
  • the gene encoding MTAN in Salmonella enterica was synthesized and cloned into pDONR221 vector by DNA 2.0 (Menlo Park, CA), incorporating an N-terminal His 6 tag sequence and terminal attB sequences.
  • the gene was swapped into pDEST14 vector (amp R ) using Invitrogen Gateway Cloning Technology (Carlsbad, CA), and successful clones were transformed into BL21 (DE3) competent cells. Several colonies were screened for protein expression under IPTG induction, and glycerol stocks were prepared from the best expression clones.
  • LB medium was seeded with an overnight culture of BL21 (DE3) grown in the presence of ampicillin (100 ⁇ g/mL), and incubated at 37 °C with shaking at 250 rpm.
  • Cells were harvested by centrifugation at 4000 rpm for 20 min and resuspended in lysis buffer (40 mM TEA-HCl pH 7.8, 300 mM NaCl, 0.2 mg/mL lysozyme, 0.1 mg/mL DNasel, protease inhibitors).
  • lysis buffer 40 mM TEA-HCl pH 7.8, 300 mM NaCl, 0.2 mg/mL lysozyme, 0.1 mg/mL DNasel, protease inhibitors.
  • Cells were lysed with three rounds of sonication for 15 s each round with 1 min cooling time between bursts.
  • Cell debris was removed by centrifugation at 16,000 rpm for 30 min, and the cleared supernatant was loaded on a 5 mL Ni-NTA column equilibrated in lysis buffer.
  • the column was washed with 5 column volumes of 10 mM imidazole in running buffer (20 mM TEA-HCl pH 7.8, 300 mM NaCl). Elution was done using a step gradient of 25 mM imidazole in running buffer with 250 mM imidazole in the final fraction. Fractions were analyzed by SDS-PAGE and pertinent fractions were pooled together and dialyzed overnight in 20 mM TEA-HCl pH 7.8, 50 mM NaCl. Glycerol was added to 10% of the volume prior to storage at -80 °C.
  • AdoHcy to ATP Conversion Buffer The 2x AdoHcy to ATP conversion buffer was prepared as previously described with minor modifications (17). In brief, 100 mL of buffer contained 100 mM Tris-acetate pH 7.7, 2 mM PEP, 2 mM sodium pyrophosphate, 2 mM PRPP, 15 mM (NH 4 ) 2 S0 4 , 15 mM (NH 4 ) 2 Mo0 4 , and phosphatase inhibitors (Roche PhosSTOP, 10 tablets).
  • the 2x conversion buffer was completed by additions to give final concentrations of 10 mM MgS0 4 , 6 U APRTase, 18 U PPD , and 250 nM MTAN and an increase in volume of 14 ⁇ .
  • Continuous assay buffer (2x) was prepared by adding 200 luciferin/luciferase (ATPlite) to 1 mL of the AdoHcy to ATP conversion buffer and warmed to room temperature.
  • AdoMet AdoMet and the DNA substrate in DNMT assay buffer (25 mM HEPES pH 7.6, 50 mM KC1, 5 % glycerol, 0.1 % DTT) in a 96-well non-binding surface microplate.
  • the mixture was incubated at room temperature for 3 min to permit any contaminating adenine, AMP, ATP or 5'-methylthioadenosine (MTA) to be converted to a stable background light signal as measured in the kinetic acquisition mode of the luminometer for 3 min.
  • DNMT was added to give a final volume of 50 ⁇ ,. After 3 min, the luminescent signal was measured for the following 3 min.
  • the AdoHcy standard curve was constructed by mixing 25 ⁇ _ , of DNMT assay buffer containing varied concentrations of AdoHcy with 25 ⁇ . of continuous assay buffer, incubating for several minutes, and then measuring the luminescence of each sample in the discontinuous acquisition mode of the luminometer. Values were background corrected using a mixture containing no AdoHcy.
  • DNMT J Rate Measurement using Discontinuous Luciferase Assay DNMTl was added to a solution of 682 nM AdoMet and 1 ⁇ g poly(dldC) in DNMT assay buffer to a concentration of 258 nM in 300 ⁇ total volume which had been equilibrated to 22 °C or 37 °C. A control reaction containing no DNMTl was also performed at 37 °C. 15 ⁇ . aliquots of the reaction were quenched with 5 ⁇ . of 100 mM HC1 at 2 min intervals from 2 to 10 min in triplicate. These were neutralized with 5 ⁇ .
  • DNMT1 Rate Measurement using Radiometric Assay The same two reactions were prepared as above for the discontinuous luciferase assay with the exception that 672 nM 5 , -adenosyl-l-[ wet iy/- 3 H]methionine was used in place of non-radioactive AdoMet. 15 ⁇ iL aliquots were removed from each reaction in triplicate at 2 min intervals from 2 to 8 min and spotted on 2.3 cm diameter Whatman DE81 filter paper circles which were allowed to dry for 30 min.
  • a standard curve was constructed by mixing 25 of the 2x continuous assay buffer with 25 AdoHcy standard solutions in a 96-well plate (Figure 2A). Luminescence measured in the continuous acquisition mode gave maximum luminescence from the AdoHcy signal within 2 min. Light output as a function of AdoHcy (pmol) gave a linear correlation ( Figure 2B). Alternatively, all solutions could be mixed in several wells and the luminescence measured after several minutes of incubation in the discontinuous acquisition mode of the luminometer to construct the same curve ( Figure 2B). The AdoHcy standard curve matched that obtained from adenine standard solutions, indicating that the coupling between AdoHcy and adenine is complete in terms of ATP yield (17). The range of this assay is broad as solutions containing 0.1-1000 pmol of AdoHcy fall within the linear range of the standard curve.
  • Human DNMTs Full-length human DNMT3a and DNMT3b are composed of 912 and 853 amino acids, respectively. The 286 amino acid C-terminal catalytic domains were expressed for kinetic analysis. These domains were modified to contain an N-terminal His 6 tag and a thrombin cleavage site. The catalytic domain was identified through sequence alignments of DNMT3a and DMNT3b from humans and mice which gave excellent homology in the C-terminal domains. All sequences contained a cysteine residue at the position for the proposed catalytic nucleophile (eg. 706 in human DNMT3a).
  • the C-terminal regions of human DNMT3a/3b also aligned well with bacterial (cytosine 5)-methyltransferases.
  • the bacterial DNMTs are smaller than mammalian DNMTs and consist primarily of the catalytic domain.
  • the catalytic domains of mouse DNMT3a and DNMT3b have been previously expressed in E. coli (19, 20).
  • CD-DNMT3a and CD-DNMT3b were transformed into strains of E. coli competent cells and it was found that BL21 star (DE3) cells afforded the highest yield of CD-DNMT3a while BL21 (DE3) pLysS cells were best for CD-DNMT3b expression. Expression was best at low temperatures (18 °C, 18 h) as much of the protein was expressed into inclusion bodies at 37 °C and could not be refolded after purification under denaturing conditions.
  • DNMT3L was also expressed since this protein has been reported to interact with DNMT3a and DNMT3b in both full-length and catalytic domain constructs and to enhance their activities (21).
  • Expression of DNMT3L from a synthetic gene construct in E. coli BL21 (DE3) cells gave protein expression after 3 h induction at 37 °C and 2.5-fold increased expression after overnight expression at 18 °C.
  • DNMT3L was purified by elution from a Ni-NTA agarose column with an imidazole gradient.
  • Human DNMT1 was also expressed. It is known to be significantly more active than DNMT3a, DNMT3b or their mixtures with DNMT3L. As the catalytic domain of DNMT1 has been reported to be inactive (22), the full-length 1620 amino acid construct was expressed in SF9 cells using a baculovirus-based expression system. Purification of DNMT 1 was also achieved by elution from a Ni column with an imidazole gradient.
  • AdoMet is unstable at neutral and alkaline pH and degrades via an intramolecular reaction giving 5'- methylthioadenosine (MTA) and homoserine lactone and a hydrolytic reaction giving adenine and S-pentosylmethionine (23).
  • MTA is a substrate for MTAN and is hydrolyzed to give adenine.
  • both breakdown pathways produce intermediates that contribute to the background in the luciferase-linked DNMT assay.
  • Commercial AdoMet sources contain up to 10 % MTA and/or adenine. HPLC purification reduced these to below 0.5 %.
  • AdoMet instability makes it necessary to measure a background
  • Bacterial M.SssI CpG methyltransferase was used to benchmark the assay because of its high activity.
  • the commonly used synthetic oligonucleotide poly(2'- deoxyinosinic-2'-deoxycytidylic acid) (poly(dldC)) was selected as the DNA substrate. It is reported to have high activity with DNMTs (24).
  • Initial reaction rates were measured with a constant amount of poly(dldC) while AdoMet concentration was varied in order to measure its K m .
  • the luminescent signal increased linearly with time as the AdoHcy product of DNA methylation was converted to ATP.
  • the rate increased with increasing AdoMet concentration ( Figure 3 A).
  • the dldC concentration of poly(dldC) was calculated based on an average polymer length of 2100 bp (1200-3000 bp range) giving an average double-stranded molecular weight of 2.57 x 10 6 g/mol and a dldC base pair concentration of 0.82 nmol dldC ⁇ g poly(dldC) (7.8 nM DNA polymer in the assay).
  • Initial reaction rates gave poor fits to the Michaelis-Menten equation since this equation includes the assumption that the substrate concentration is unchanged by enzyme binding and is constant during the course of the reaction.
  • corrections can be made by fitting to the Morrison equation for substrate depletion (25):
  • the AdoMet K m for CD-DNMT3 a-DNMT3 L is more than 70-fold lower than that for M.Sssl CpG methyltransferase.
  • Both human CD- DNMT3-DNMT3L mixtures have k cat values two to three orders of magnitude lower than that for the M.Sssl CpG methyltransferase.
  • Full-length human DNMTl prefers hemimethylated DNA as a substrate in which one of the two strands contains methylated cytosine, however, it is more active on unmethylated poly(dldC) DNA than CD-DNMT3a or CD-DNMT3b in complex with
  • DNMT3L (24). DNMTl was used at a concentration of 516 nM with while varying AdoMet and DNA concentrations ( Figures 4D and 4E). Initial rate data were fit to the Morrison equation to give a AdoMet K m of 2.7 ⁇ 0.7 ⁇ , a poly(dldC) K m of 840 ⁇ 400 nM, and a k cal of 28 ⁇ 3 h "1 (Table 1). These values are similar to those for the bacterial M.Sssl CpG methyltransferase, although its k cat value is 6.5 times greater than that for human DNMTl , consistent with reported values (6, 26). This difference between DNMTl and the CD- DNMT3 a-DNMT3 L and CD-DNMT3b-DNMT3L complexes suggest the need for additional regulatory elements for activation of the human DNMT3 sequences.
  • Discontinuous assays have applications in endpoint assays and inhibitor screens. They are also suited to the
  • a light-based continuous detection assay has been developed for the detection of AdoHcy.
  • the assay will provide facile quantitation of any AdoMet-based
  • methyltransferases because of the sensitivity, dynamic range and link to light production with recycling of AMP for stable light signal.
  • the range of applications is broadened by use under both continuous and discontinuous conditions, as needed for chemical library screening and hit validation.
  • dldC base pair concentration 16.4 ⁇ .
  • AdoMet concentration 35 ⁇ for DNMT1 , 10 ⁇ for CD- DNMT3a and CD-DNMT3b in complexes with DNMT3L.

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Abstract

La présente invention concerne des méthodes de détection de l'activité de l'ADN (cytosine 5)-méthyltransférase, de la protéine méthyltransférase ou de toute enzyme formant en tant que produit la S-adénosyl-1-homocystéine (AdoHcy), ainsi que de recherche par criblage d'inhibiteurs d'ADN (cytosine 5)-méthyltransférase, de protéine méthyltransférase et de toute enzyme formant en tant que produit la S-adénosyl-1-homocystéine (AdoHcy) à l'aide de dosages de liaison à la luciférase convertissant le produit S-adénosyl-1-homocystéine (AdoHcy) en un signal luminescent quantifiable.
PCT/US2011/000430 2010-03-10 2011-03-08 Analyse par liaison à la luciférase de l'adn-méthyltransférase, de la protéine méthyltransférase et de la s-adénosylhomocystéine et leurs applications WO2011112245A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013112358A1 (fr) * 2012-01-23 2013-08-01 Albert Einstein College Of Medicine Of Yeshiva University Procédés par liaison à la luciférase de détection de l'adénosine et utilisations correspondantes
CN105586385A (zh) * 2016-03-10 2016-05-18 北京中科唯新生物医学研究所有限公司 一种磷脂酰乙醇胺n-甲基转移酶酶活力检测方法
CN105695558A (zh) * 2016-03-10 2016-06-22 北京中科唯新生物医学研究所有限公司 一种磷脂酰乙醇胺n-甲基转移酶酶活力检测试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891659A (en) * 1996-03-04 1999-04-06 Kikkoman Corporation Bioluminescent adenosine phosphate ester assay and reagent
WO2007087541A2 (fr) * 2006-01-23 2007-08-02 Washington State University Research Foundation Dosages pour méthyltransférases dépendantes de la s-adénosylméthionine
US20070224655A1 (en) * 2006-03-24 2007-09-27 Regents Of The University Of Michigan Methyltransferase assays

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GB2392911A (en) * 2001-05-11 2004-03-17 Methylgene Inc Inhibitors of dna methyltransferase isoforms

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891659A (en) * 1996-03-04 1999-04-06 Kikkoman Corporation Bioluminescent adenosine phosphate ester assay and reagent
WO2007087541A2 (fr) * 2006-01-23 2007-08-02 Washington State University Research Foundation Dosages pour méthyltransférases dépendantes de la s-adénosylméthionine
US20070224655A1 (en) * 2006-03-24 2007-09-27 Regents Of The University Of Michigan Methyltransferase assays

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013112358A1 (fr) * 2012-01-23 2013-08-01 Albert Einstein College Of Medicine Of Yeshiva University Procédés par liaison à la luciférase de détection de l'adénosine et utilisations correspondantes
CN105586385A (zh) * 2016-03-10 2016-05-18 北京中科唯新生物医学研究所有限公司 一种磷脂酰乙醇胺n-甲基转移酶酶活力检测方法
CN105695558A (zh) * 2016-03-10 2016-06-22 北京中科唯新生物医学研究所有限公司 一种磷脂酰乙醇胺n-甲基转移酶酶活力检测试剂盒

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